The present study investigated whether alterations in the muscle high energy phosphate state initiates the contraction-induced increase in skeletal muscle GLUT4 protein concentration. Sprague-Dawley rats were provided either a normal or a 2% beta-guanidinoproprionic acid (beta-GPA) diet for 8 weeks and then the gastrocnemius of one hind limb was subjected to 0, 14 or 28 days of chronic (24 h day-1) low-frequency electrical stimulation (10 Hz). The beta-GPA diet, in the absence of electrical stimulation, significantly reduced ATP, creatine phosphate, creatine and inorganic phosphate and elevated GLUT4 protein concentration by 60% without altering adenylate cyclase activity or cAMP concentration. Following 14 days of electrical stimulation, GLUT4 protein concentration was elevated above non-stimulated muscle in both groups but was significantly more elevated in the beta-GPA group. Concurrent with this greater rise in GLUT4 protein concentration was a greater decline in the high energy phosphates and a greater rise in cAMP. After 28 days of electrical stimulation, GLUT4 protein concentration and cAMP stabilized and was not different between diet treatments. However, the high energy phosphates were significantly higher in the normal diet rats as opposed to the beta-GPA rats. These findings therefore suggest that a reduction in cellular energy supply initiates the contraction-induced increase in muscle GLUT4 protein concentration, but that a rise in cAMP may potentiate this effect.